M. bovis is a bovine pathogen in housed or intensively reared beef and dairy cattle. The most frequently reported clinical manifestation is pneumonia of calves, which is often accompanied by arthritis, also known as pneumonia-arthritis syndrome. Its etiological role has also been associated with mastitis, otitis, and reproductive disease or disorders of cows and bulls. Significant economic losses are linked with M. bovis induced respiratory disease, since M. bovis has been associated with up to 36% of the mortality due to bovine respiratory disease (BRD). In order to reduce mortality, antibiotic therapy is often used since no fully licensed vaccines are currently available. Prevention of M. bovis disease may also reduce predisposition of the animal to other respiratory diseases. Therefore, there is a need to develop efficacious and safe vaccines against M. bovis. M. hyo is a bacterial pathogen that causes enzootic pneumonia in swine. The majority of known vaccines against M. hyo are based on inactivated whole cell preparations of M. hyo. Other vaccines against M. hyo include subunit vaccines composed of M. hyo derived proteins, polypeptides or immunogenic fragments of such proteins or polypeptides, and DNA vaccines composed of DNA encoding for one or more M. hyo derived proteins or polypeptides and immunogenic fragments thereof.
Examples of whole cell inactivated M. hyo vaccines include chemically inactivated whole cell cultures with M. hyo, coupled with an oil adjuvant (RESPISURE® and STELLAMUNE™) commercially available from Pfizer Inc., USA.
A number of M. hyo proteins have been described. International Patent Publication WO 96/28472 describes six protein antigen species of M. hyo at molecular weights of 46-48, 52-54, 60-64, 72-75, 90-94 and 110-114 kilodaltons, and discloses partial protein sequences of the 52-54, 60-64 and 72-75 kilodalton antigens and the full length nucleotide and amino acid sequences of the 46-48 kilodalton antigen.
The cloning of the gene encoding the M. hyo protein P46, i.e. p46, was also described by Futo et al. in J. Bacteriol 177:1915-1917(1995) and further in European Patent Publication No. 0 475 185 A1.
Wise and Kim (1987, J. Bacteriol. 169: 5546-5555) reported four integral membrane protein species in M. hyo, named p70, p65 (P65, supra), p50 and p44, the latter three of which are modified by covalent lipid attachments and induce a strong humoral immune response. The protective effects of the immune response were not investigated. The gene encoding the P65 protein has been cloned, and its sequences and uses in vaccines and diagnostics are described in U.S. Pat. No. 5,788,962.
International Patent Publication WO 91/15593 describes five proteins of M. hyo of apparent molecular weights of 105, 90, 85, 70 and 43 kilodaltons. A full-length sequence of the gene encoding 85 kilodalton protein (protein C) was provided, as were partial nucleotide sequences encoding the other four proteins.
U.S. Pat. No. 5,252,328 to Faulds discloses amino terminal sequences of immunoreactive M. hyo proteins, the molecular weights of which are 36, 41, 44, 48, 64, 68, 74.5, 79, 88.5, 96 and 121 kilodaltons. Other proteins identified based on the electrophoretic mobilities but for which no protein sequences were disclosed had apparent molecular weights of 22.5, 34 and 52 kilodaltons. While U.S. Pat. No. 5,252,328 proposed the use of these proteins in vaccine formulations against M. hyo infections, no results of vaccine trials were reported.
International Patent Publication WO 95/09870 discloses biochemical methods for the purification of M. hyo adhesins, the mycoplasmal integral membrane proteins responsible for adhesion to the cilia of the host's upper respiratory epithelium. WO 95/09870 also proposes assays and uses for these proteins, for example in vaccines and diagnostics.
A research paper by King et al. (1997; Vaccine 15:25-35) disclosed Mhp1, a 124 kilodalton adhesin that is a strain variant of P97.
A 94 kilodalton variant of P97 was identified by Wilton et al. (1998, Microbiology 144:1931-1943). Additionally, the p97 gene was shown to be part of an operon that also encodes a second protein, termed P102, of a predicted molecular weight of approximately 102 kilodaltons (Hsu et al., 1998, Gene 214:13-23). Minion and Hsu suggest the use of P102 in vaccines against M. hyo infections in the international patent publication WO 99/26664 but do not report vaccine trials.
Prior to the present invention, there has been no recognition that a M. hyo vaccine can provide protective effects in cattle against disorders caused by M. bovis. 